phalloidine and phallacidin

Most of the fatal cases of mushroom poisoning are caused by Amanita phalloides. The amount of toxin in mushroom varies according to climate and environmental conditions. The aim of this study is to measure α-, β-, and γ-amanitin with phalloidin and phallacidin toxin concentrations. Six pieces of A. phalloides mushrooms were gathered from a wooded area of Düzce, Turkey, on November 23, 2011. The mushrooms were broken into pieces as spores, mycelium, pileus, gills, stipe, and volva. α-, β-, and γ-Amanitin with phalloidin and phallacidin were analyzed using reversed-phase high-performance liquid chromatography. As a mobile phase, 50 mM ammonium acetate + acetonitrile (90 + 10, v/v) was used with a flow rate of 1 mL/min. C18 reverse phase column (150 × 4.6 mm; 5 µm particle) was used. The least amount of γ-amanitin toxins was found at the mycelium. The other toxins found to be in the least amount turned out to be the ones at the spores. The maximum amounts of amatoxins and phallotoxin were found at gills and pileus, respectively. In this study, the amount of toxin in the spores of A. phalloides was published for the first time, and this study is pioneering to deal with the amount of toxin in mushrooms grown in Turkey.

Topics: Alpha-Amanitin; Amanita; Amanitins; Chromatography, High Pressure Liquid; Chromatography, Reverse-Phase; Forests; Fruiting Bodies, Fungal; Humans; Mushroom Poisoning; Mycelium; Peptides, Cyclic; Phalloidine; Species Specificity; Spectrophotometry, Ultraviolet; Spores, Fungal; Turkey

Accidental or deliberate poisoning of food is of great national and international concern. Detecting and identifying potentially toxic agents in food is challenging due to their large chemical diversity and the complexity range of food matrices. A methodology is presented whereby toxic agents are identified and further characterized using a two-step approach. First, generic screening is performed by LC/MS/MS to detect toxins based on a list of selected potential chemical threat agents (CTAs). After identifying the CTAs, a second LC/MS analysis is performed applying accurate mass determination and the generation of an attribution profile. To demonstrate the potential of the methodology, toxins from the mushrooms Amanita phalloides and Amanita virosa were analyzed. These mushrooms are known to produce cyclic peptide toxins, which can be grouped into amatoxins, phallotoxins and virotoxins, where α-amanitin and β-amanitin are regarded as the most potent. To represent a typical complex food sample, mushroom stews containing either A. phalloides or A. virosa were prepared. By combining the screening method with accurate mass analysis, the attribution profile for the identified toxins and related components in each stew was established and used to identify the mushroom species in question. In addition, the analytical data was consistent with the fact that the A. virosa specimens used in this study were of European origin. This adds an important piece of information that enables geographic attribution and strengthens the attribution profile.

Topics: Amanita; Amanitins; Chromatography, Liquid; Humans; Mass Spectrometry; Mushroom Poisoning; Peptides, Cyclic; Phalloidine; Poisons

The identification of toxic oligopeptides employing CE-ESI-MS is presented. The analytes studied ama- and phallotoxins are of significant forensic interest because over 90% of the lethal cases of fungus poisoning in man are caused by species of Amanita which contain these toxins. A CE method was developed to separate the toxins alpha-, beta- and gamma-amanitin, phalloidin and phallacidin. Their fragmentation patterns in MS(n) experiments were investigated in the positive and in the negative ion mode, also the influence of the sheath liquid mixture of the used interface on the S/N. Method validation included the determination of the LOD and the repeatability of the migration time and peak area for both detection modes. With the optimized method LODs of 13-79 ng/mL (17-87 nmol/L) were reached. The CE-MS procedure was successfully applied to the identification of ama- and phallotoxins in extracts of air-dried mushroom samples.

Topics: Amanita; Amanitins; Electrophoresis, Capillary; Humans; Molecular Structure; Mushroom Poisoning; Mycotoxins; Oligopeptides; Peptides, Cyclic; Phalloidine; Spectrometry, Mass, Electrospray Ionization

Although changes of cytoskeleton (CSK) stiffness and friction can be induced by diverse interventions, all mechanical changes reported to date can be scaled onto master relationships that appear to be universal. To assess the limits of the applicability of those master relationships, we focused in the present study on actin and used a panel of actin-manipulating drugs that is much wider than any used previously. We focused on the cultured rat airway smooth muscle (ASM) cell as a model system. Cells were treated with agents that directly modulate the polymerization (jasplakinolide, cytochalasin D, and latrunculin A), branching (genistein), and cross linking (phallacidin and phalloidin oleate) of the actin lattice. Contractile (serotonin, 5-HT) and relaxing (dibutyryl adenosine 3',5'-cyclic monophosphate, DBcAMP) agonists and a myosin inhibitor (ML-7) were also tested for comparison, because these agents may change the structure of actin indirectly. Using optical magnetic twisting cytometry, we measured elastic and frictional moduli before and after treatment with each agent. Stiffness increased with frequency as a weak power law, and changes of friction paralleled those of stiffness until they approached a Newtonian viscous limit. Despite large differences in the mechanism of action among the interventions, all data collapsed onto master curves that depended on a single parameter. In the context of soft glassy systems, that parameter would correspond to an effective temperature of the cytoskeletal matrix and reflect the effects of molecular crowding and associated molecular trapping. These master relationships demonstrate that when the mechanical properties of the cell change, they are constrained to do so along a special trajectory. Because mechanical characteristics of the cell shadow underlying molecular events, these results imply special constraints on the protein-protein interactions that dominate CSK mechanical properties.

Topics: Actins; Animals; Azepines; Biomechanical Phenomena; Bridged Bicyclo Compounds, Heterocyclic; Bucladesine; Cells, Cultured; Cross-Linking Reagents; Cytochalasin D; Genistein; Muscle Contraction; Myocytes, Smooth Muscle; Myosins; Naphthalenes; Peptides, Cyclic; Phalloidine; Rats; Rheology; Serotonin; Serotonin Antagonists; Serotonin Receptor Agonists; Thiazoles; Thiazolidines; Trachea

Effects on morphology and microfilament structure caused by phalloidin, phallacidin, and some semisynthetic phalloidin derivatives were studied in vegetative cells of the green alga Acetabularia acetabulum (L.) Silva. All phalloidin derivatives (except for phalloidin itself) caused growth stop of the alga after 1 day and (except for the fluorescein-labeled phalloidin) death of the cells after 4-7 days. Hair whorl tip growth and morphology as screened by light microscopy, as well as microfilament structure in tips, suggested that growth stop is correlated with a disorganization of actin filaments similar to that recently described for jasplakinolide (H. Sawitzky, S. Liebe, J. Willingale-Theune, D. Menzel, European Journal of Cell Biology 78: 424-433, 1999). Using rabbit muscle actin as a model target protein, we found that the toxic effects in vivo did not correlate with actin affinity values, suggesting that permeation through membranes must play a role. Indeed, the most lipophilic phalloidin derivatives benzoylphalloidin and dithiolanophalloidin were the most active in causing growth stop at ca. 100 microM. In comparison to the concentration of jasplakinolide required to cause similar effects (<3 microM), the two most active phalloidin derivatives exhibited an activity ca. 30 times lower. Nonetheless, lipophilic phalloidin derivatives can be used in algae, and probably also other cells, to modulate actin dynamics in vivo. In addition, we found that the fluorescent fluorescein isothiocyanate-phalloidin is able to enter living algal cells and stains actin structures brightly. Since it does not suppress actin dynamics, we suggest fluorescein isothiocyanate-phalloidin as a tool for studying rearrangements of actin structures in live cells, e.g., by confocal laser scanning microscopy.

Topics: Acetabularia; Actin Cytoskeleton; Actins; Fluorescent Dyes; Hydrogen-Ion Concentration; Microscopy, Confocal; Peptides, Cyclic; Phalloidine

The toxin composition of 25 Amanita phalloides carpophores collected from three sites in Franche-Comté (France) differing in their geological and pedological characteristics was determined and the factors involved in the variations of the toxin concentration in the tissues were identified. The concentrations of the main amatoxins (beta-amanitin, alpha-amanitin, gamma-amanitin) and phallotoxins (phallacidin, phallisacin, phalloidin, phallisin, phalloin) in the six tissues constituting the carpophore, i.e. the cap (C), gills (G), ring (R), stipe (S), bulb (B) and volva (V) were evaluated by using high-performance liquid chromatography. The results analysed statistically showed that the toxin concentrations were tissue dependent, leading to classification of the tissues into two groups (B, V) and (C, G, R, S). The (B, V) group was distinguished by high amounts of phalloidin, phallisin and phallisacin, and the (C, G, R, S) group by the predominance of the amatoxins. The characteristics of the soil of the collection site also affected the toxin concentrations; however, this effect differed from one site to another and was not similar for all the tissues. Finally, the mean toxin profile in the carpophores from the three sites was evaluated. This study underscores the fact that environmental factors and mainly the soil type clearly have an effect on the toxin composition of A. phalloides carpophores.

Topics: Alkaloids; Amanita; Amanitins; Environment; France; Hydrogen-Ion Concentration; Peptides, Cyclic; Phalloidine; Soil

Frozen sections of the rat and mouse testes were stained with either FITC-phalloidin or NBD-phallacidin and viewed with conventional fluorescence and confocal laser microscopes in order to demonstrate the arrangement of actin-filament bundles in myoid cells, Sertoli cells and tunica albuginea. Myoid cells are rich in actin-filament bundles crossing at right angles. These bundles running in different directions can also be visualized by means of electron microscopy. Nerve fibers occur in the vicinity of myoid cells, suggesting a neural control of the cell. At Sertoli cell junctions actin filaments occur at the circumference of the cell, where they show a honeycomb pattern. The ratio of the number of Sertoli cells per myoid cell can be calculated by means of confocal microscopy; this technique may provide a new parameter for determining spermatogenic activity. In the tunica albuginea of the juvenile mouse testis, actin filaments are arranged in an alternate fashion.

Topics: Actins; Animals; Connective Tissue; Connective Tissue Cells; Image Processing, Computer-Assisted; Lasers; Male; Mice; Mice, Inbred BALB C; Peptides, Cyclic; Phalloidine; Rats; Rats, Inbred Strains; Sertoli Cells; Spermatogenesis; Testis

Primary cultures of rat hepatocytes were used for assaying several drugs not previously known for inhibiting the transport of phalloidin. In order to have 50% inhibition (IC50) of the entrance of a tritiated phallotoxin derivative ([3H]demethylphalloin, 1 microM) from the medium into the cells the following concentrations (microM) of the various inhibitors were determined: cyclolinopeptide (0.5), Nocloprost (5.0), Nileprost (7.0), beta-estradiol (42), Verapamil (70). For comparison, the corresponding IC50 values of some known antagonists of phalloidin toxicity were determined by the same method. Moreover, we studied several natural and synthetic phallotoxins and alpha-amanitin for their ability to displace [3H]demethylphalloin from the transporting system. Lineweaver-Burk plots made it obvious that two groups of inhibitors exist. Competitive inhibitors are, for example, antamanide, beta-estradiol, silybin, Nileprost, taurocholate, and the cyclic somatostatin analog cyclo[Phe-Thr-Lys-Trp-Phe-D-Pro], whereas Verapamil and monensin inhibit phallotoxin uptake in a non-competitive way. Considering the very different chemical features of the competitive inhibitors, we tentatively conclude that the phallotoxin transport system selects compounds not on the basis of their chemical features, but rather their physical properties. The physical properties of a typical substrate are low molecular mass, lipophilic nature, and, possibly the presence of rigid ring structures. Negative charges accelerate the transport of a substrate, while positive charges have the opposite effect. The phalloidin-transporting system may represent part of a hepatic equipment which clears portal blood from, for example, bile acids, lipophilic hormones, or xenobiotics. By chance, the transporting system incorporates phallotoxins into the hepatocytes leading to the death of these cells.

Topics: Amanitins; Animals; Binding, Competitive; Biological Transport; Cells, Cultured; Cholic Acids; Epoprostenol; Estradiol; Kinetics; Liver; Monensin; Oligopeptides; Peptides, Cyclic; Phalloidine; Prostaglandins F, Synthetic; Rats; Silybin; Silymarin; Somatostatin; Taurocholic Acid; Verapamil